1. Field of the Invention
The present invention generally relates to particle size analysis and, further, to analysis of biological particles for diagnostic purposes utilizing traditional particulate size or mobility measurement devices. One aspect of the present invention more particularly relates to medical diagnostics for the quantitative and qualitative analysis of lipoprotein classes and subclasses and their relationship to the assignment of coronary heart disease and other lipid-related health risks.
2. Description of the Relevant Art
Introduction
In clinical practice, lipoprotein particle measurements are used to assess cardiovascular and other lipid-related health risks, determine treatment protocols and track the efficacy of treatment regimens. Lipoprotein particles comprise macromolecules that package cholesterol and other biochemicals, enabling them to be transported through the blood stream. The size distribution of lipoprotein particles varies among individuals due to both genetic and nongenetic influences. The diameters of lipoprotein particles typically range from about 7 nm to about 120 nm. In this diameter size range, there exist subfractions of the particles that are important predictors of cardiovascular disease. For instance, very low density lipoproteins transport triglycerides in the blood stream; high very low density lipoprotein levels in the blood stream are indicative of hypertriglyceremia. These subfractions are identified by analytical techniques that display the quantity of material as a function of lipoprotein particle size or density.
Standard Plasma Lipid and Lipoprotein Cholesterol Measurement Techniques
Typical standard lipid measurements include fasting total cholesterol, triglyceride, as well as HDL and LDL cholesterol. Currently, the most widely used method for measuring LDL cholesterol is the indirect Friedewald method (Friedewald, et al., Clin. Chem. Vol. 18, pp. 499-502, 1972). The Friedewald assay method requires three steps: 1) determination of plasma triglyceride (TG) and total cholesterol (TC), 2) precipitation of VLDL and LDL, and 3) quantitation of HDL cholesterol (HDLC). Using an estimate for VLDLC as one-fifth of plasma triglycerides
      (                  T        ⁢                                  ⁢        G            5        )    ,the LDL cholesterol concentration (LDLC) is calculated by the formula: LDLC=TC−(HDLC+VLDLC). While generally useful, the Friedewald method is limited in its accuracy in specific cases. Errors can occur in any of the three steps, in part because this method requires that different procedures be used in each step. The Friedewald method is to a degree indirect, as it presumes that VLDLC concentration is one-fifth that of plasma triglycerides. When the VLDL of some patients deviates from this ratio, further inaccuracies occur. Ultracentrifugation must be employed for separation and subsequent determination of LDL cholesterol for some samples, since the Friedewald method cannot be used for patients with TG over 400 mg/dL.
Procedures for Lipoprotein Subspecies Analysis
Presently, the predominant methods for lipoprotein subspecies analysis include nuclear magnetic resonance, the vertical auto profile, and Electrophoretic gel separation. Each of these methods will be briefly discussed below.
Nuclear Magnetic Resonance
Otvos teaches a nuclear magnetic resonance (NMR) procedure for determining the concentrations of lipoprotein subclasses, which has greater accuracy than Friedewald (U.S. Pat. No. 5,343,389, issued Aug. 30, 1994). Otvos initially obtains the NMR chemical shift spectrum of a blood plasma or serum sample. The observed spectrum of the entire plasma sample is then matched with the known weighted sums of NMR spectra of lipoprotein subclasses, which are stored in a computer software program. The weight factors that give the best fit between the sample spectrum and the calculated spectrum are then used to estimate the concentrations of constituent lipoprotein subclasses in the blood sample. This procedure has the additional advantage of being rapid.
Vertical Auto Profile
Another lipoprotein subfraction determination method that is used clinically is the Vertical Auto Profile (VAP), (Kulkarni, et al., J. Lip. Res., Vol. 35, pp. 159-168, 1994). In the Vertical Auto Profile method, a flow analyzer is used for the enzymatic analysis of cholesterol in lipoprotein classes separated by a short spin single vertical ultracentrifugation, with subsequent spectrophotometry and software analysis of the resulting data. While a useful advance, this technique does not resolve the LDL into all seven subspecies identified by electrophoretic gradient gel separation.
Electrophoretic Gradient Gel Separation
The gel separation method is demonstrated in U.S. Pat. No. 5,925,229, issued Jul. 20, 1999, by R. M. Krauss, et al., entitled “Low Density Lipoprotein Fraction Assay for Cardiac Disease Risk,” (the '229 patent), which is hereby incorporated by reference. In the '229 patent, a gradient gel electrophoresis procedure for the separation of LDL subclasses is disclosed. The LDL fractions are separated by gradient gel electrophoresis, producing results that are comparable to those obtained by ultracentrifuge. This method generates a fine resolution of LDL subclasses, and is used principally by research laboratories. However, gradient gel electrophoresis can take many hours to complete. It would be useful if gradient gel electrophoresis separation times could be shortened and the analysis simplified so that high resolution lipid analysis could be used in clinical laboratories as part of a routine screening of blood samples, and to assign a risk factor for coronary artery disease.
The gel separation method, which depends on uniform staining of all components that are subsequently optically measured, suffers from nonuniform chromogenicity. That is, not all lipoprotein particles stain equally well. The differential stain uptake produces erroneous quantitative results, in that a less staining peak may be read at a lower value than is actually present. Additionally, the nonuniform chromogenicity can result in erroneous qualitative results, in that measured peaks may be skewed to a sufficient degree as to confuse one class or subclass of lipoprotein with another.
A high-resolution assay for measuring all subclasses of LDL as well as VLDL, IDL, HDL, and chylomicron particles that would be accurate, direct, and complete, would be an important innovation in lipid measurement technology. If inexpensive and convenient, such an assay could be employed not only in research laboratories, but also in a clinical laboratory setting. Ideally, clinicians could use this information to improve current estimation of coronary disease risk and make appropriate medical risk management decisions based on the assay.